Coronavirus vaccines comprising a tlr9 agonist

ABSTRACT

The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Application No. 62/983,737, filed Mar. 1, 2020, the disclosure of which is incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 377882007840SEQLIST.TXT, date recorded: Feb. 27, 2021, size: 23 KB).

FIELD

The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

BACKGROUND

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Initial symptoms of COVID-19, also known as Wuhan pneumonia, include one or more of fever, cough, and shortness of breath appearing within about 2-14 days of exposure to SARS-CoV-2. Although most cases of COVID-10 are mild, nearly 5% progress to respiratory failure, septic shock and/or multiple organ failure, with a case fatality rate of about 2.3% (Wu and McGoogan, JAMA, 323(13):1239-1242, 2020).

SARS-CoV-2 is spread through contact with respiratory droplets produced when an infected person coughs or exhales. According to the World Health Organization (WHO), as of Mar. 1, 2020 there are over 85,000 confirmed COVID-19 cases in 60 countries leading WHO to declare the current outbreak as a public health emergency of international concern. According to the worldometer, nearly one year later there are over 110 million coronavirus cases accounting for over 2.5 million deaths worldwide, with over 29 million coronaviruses cases accounting for over 500,000 deaths in the United States alone. In order to prevent person-to-person transmission of SARS-CoV-2, basic measures such as frequently washing hands, avoidance of touching eyes, nose and mouth, and an avoiding travel and public activities are recommended.

However, to reduce the risk of SARS-CoV-2 infection without curtailing everyday activities, a COVID-19 vaccine is needed. In particular, a COVID-19 vaccine that is able to rapidly induce an immune response against SARS-CoV-2 is urgently needed.

SUMMARY

The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows induction of neutralizing antibodies by CpG 1018 and aluminum hydroxide-adjuvanted SARS-CoV-2 S-2P at 2 weeks post-second injection. BALB/c mice (N=6 per group) were immunized with 2 dose levels of Chinese hamster ovary (CHO) cell-expressed SARS-CoV-2 S-2P adjuvanted with CpG 1018, aluminum hydroxide or a combination of both 3 weeks apart and the antisera were harvested at 2 weeks after the second injection. The antisera were subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to determine the ID₅₀(left) and ID₉₀(right) titers of neutralization antibodies.

FIG. 2 shows total anti-S IgG titers in mice immunized with S-2P with adjuvants. Sera from BALB/c mice in FIG. 1 (N=6 per group) immunized with 0, 1 or 5 μg of S-2P with CpG 1018, aluminum hydroxide or a combination of both were quantified for the total amount of anti-S IgG in an enzyme linked immunosorbent assay (ELISA).

FIG. 3 shows neutralization of wild-type SARS-CoV-2 virus by antibodies induced by SARS-CoV-2 S-2P adjuvanted with CpG 1018 and aluminum hydroxide. The antisera were collected as described in FIG. 2 (N=6 per group) and subjected to a neutralization assay with wild-type SARS-CoV-2 to determine neutralization antibody titers.

FIG. 4 shows inhibition of pseudoviruses carrying D614D (wild-type) or D614G (variant) versions of the spike protein by mice immunized with S-2P with CpG 1018 and aluminum hydroxide. The antisera of BALB/c mice immunized with 1 or 5 μg of S-2P with 10 μg CpG 1018 and 50 μg aluminum hydroxide as in FIG. 1 (N=5 per group due to assay capacity) were collected. Neutralization assays were performed with pseudoviruses with either D616D or D614G spike proteins.

FIG. 5 shows IFN-γ/IL-4, IFN-γ/IL-5, and IFN-γ/IL-6 ratios. IFN-γ, IL-4, IL-, and IL-6 values from the cytokine assays (N=6 per group) were used to calculate ratios. Ratio values greater than 1 indicate Th1 bias whereas ratio less than 1 indicate Th2 bias responses.

FIGS. 6A-6B show neutralizing antibody titers with pseudovirus assay in hamsters 2 weeks after second immunization. Hamsters (N=10 per group) were immunized twice at 3 weeks apart with vehicle control (PBS), 1 μg (LD) or 5 μg (HD) of S-2P adjuvanted with 150 μg CpG 1018 and 75 μg aluminum hydroxide, or with adjuvant alone. The antisera were harvested at 2 weeks after the second injection and subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to determine the ID₉₀ titers of neutralization antibodies (FIG. 6A) and total anti-S IgG antibody titers with ELISA (FIG. 6B). Results are presented as geometric mean with error bars representing 95% confidence interval and statistical significance calculated with Kruskal-Wallis with corrected Dunn's multiple comparisons test. Dotted lines represent lower and upper limits of detection (40 and 5120 in ID₉₀, 100 and 1,638,400 in IgG ELISA).

FIGS. 7A-7B show viral load in hamsters 3 or 6 days post infection (dpi) with SARS-CoV-2. The hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected for viral load determination by quantitative PCR of viral genome RNA (FIG. 7A), and TCID₅₀ assay for virus titer (FIG. 7B). Results are presented as geometric mean with error bars representing 95% confidence interval and statistical significance calculated with Kruskal-Wallis with corrected Dunn's multiple comparisons test. Dotted lines represent lower and limit of detection (100).

FIG. 8 shows lung pathology scoring in hamsters 3 or 6 days post infection (dpi) with SARS-CoV-2. The hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected for sectioning and staining. The histopathology sections were scored as outlined in the methods and the results tabulated. Results are presented as mean of lung pathology scores with error bars representing standard error and statistical significance calculated with one-way ANOVA with Tukey's multiple comparisons test.

GENERAL TECHNIQUES AND DEFINITIONS

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., about 3000 μg of CpG 1018 refers to 2700 μg to 3300 μg of CpG 1018).

As used interchangeably herein, the terms “polynucleotide” and “oligonucleotide” include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides or combinations thereof. The oligonucleotide can be linearly or circularly configured, or the oligonucleotide can contain both linear and circular segments. Oligonucleotides are polymers of nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such as phosphorothioate esters may also be used in oligonucleotides. A nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. A nucleotide is a phosphate ester of a nucleoside.

The terms “CpG”, “CpG motif,” and “cytosine-phosphate-guanosine,” as used herein, refer to an unmethylated cytidine-phospho-guanosine dinucleotide, which when present in an oligonucleotide contributes to a measurable immune response in vitro, in vivo and/or ex vivo. Examples of measurable immune responses include, but are not limited to, antigen-specific antibody production, secretion of cytokines, activation or expansion of lymphocyte populations, such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and the like. Preferably, the CpG oligonucleotide preferentially activates a Th1-type response.

An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering an immunogenic composition, an effective amount contains sufficient antigen and TLR9 agonist to stimulate an immune response (preferably a seroprotective level of antibody to the antigen).

The terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats) and pets (e.g., dogs and cats).

The term “dose” as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.

The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). The term “isolated,” when used in reference to a recombinant protein, refers to a protein that has been removed from the culture medium of the host cell that produced the protein.

“Stimulation” of a response or parameter includes eliciting and/or enhancing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., increase in TLR-signaling in the presence of a TLR agonist as compared to the absence of the TLR agonist). For example, “stimulation” of an immune response means an increase in the response. Depending upon the parameter measured, the increase may be from 5-fold to 500-fold or over, or from 5, 10, 50, or 100-fold to 500, 1,000, 5,000, or 10,000-fold.

As used herein the term “immunization” refers to a process that increases a mammalian subject's reaction to antigen and therefore improves its ability to resist or overcome infection.

The term “vaccination” as used herein refers to the introduction of vaccine into a body of a mammalian subject.

“Adjuvant” refers to a substance which, when added to a composition comprising an antigen, nonspecifically enhances or potentiates an immune response to the antigen in the recipient upon exposure.

DETAILED DESCRIPTION

The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

I. Immunogenic Compositions and Kits

The present disclosure relates to immunogenic compositions for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of from 8 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (also referred to as CpG or cytosine-phosphate-guanosine) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof.

A. Toll-Like Receptor 9 (TLR9) Agonists

Toll-like receptors (TLRs) are expressed on dendritic cells and other innate immune cells and are among the most important receptors for stimulating a response to the presence of invading pathogens. Humans have multiple types of TLRs that are similar in structure but recognize different parts of viruses or bacteria. By activating specific TLRs, it is possible to stimulate and control specific types of innate immune responses that can be harnessed to enhance adaptive responses.

TLR9 (CD289) recognizes unmethylated cytidine-phospho-guanosine (CpG) motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and to stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Based on structure and biological function, CpG-ODNs have been divided into three general classes: CpG-A, CpG-B, and CpG-C(Campbell, Methods Mol Biol, 1494:15-27, 2017). The degree of B cell activation varies between the classes with CpG-A ODNs being weak, CpG-C ODNs being good, and CpG-B ODNs being strong B cell activators. Oligonucleotide TLR9 agonists of the present disclosure are preferably good B cell activators (CpG-C ODN) or more preferably strong (CpG-B ODN) B cell activators.

Optimal oligonucleotide TLR9 agonists often contain a palindromic sequence following the general formula of: 5′-purine-purine-CG-pyrimidine-pyrimidine-3′, or 5′-purine-purine-CG-pyrimidine-pyrimidine-CG-3′ (U.S. Pat. No. 6,589,940). TLR9 agonism is also observed with certain non-palindromic CpG-enriched phosphorothioate oligonucleotides, but may be affected by changes in the nucleotide sequence. Additionally, TLR9 agonism is abolished by methylation of the cytosine within the CpG dinucleotide. Accordingly in some embodiments, the TLR9 agonist is an oligonucleotide of from 8 to 35 nucleotides in length comprising the sequence 5′-AACGTTCG-3′. In some embodiments, the oligonucleotide is greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length. In some embodiments, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising the sequence 5′-AACGTTCGAG-3′ (SEQ ID NO:3). In some embodiments, the oligonucleotide is greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length.

Researchers at Dynavax Technologies Corporation (Emeryville, Calif.) have identified a 22-mer phosphorothioate linked oligodeoxynucleotide, CpG 1018, which contains specific sequences that can substantially enhance the immune response to co-administered antigens across species (Campbell, Methods Mol Biol, 1494:15-27, 2017). CpG 1018 (5′-TGACTGTGAA CGTTCGAGAT GA-3′, set forth as SEQ ID NO:1) was chosen after screening a broad panel of oligonucleotides for immunostimulatory activity in vitro and in vivo. CpG 1018 is a CpG-B ODN that is active in mice, rabbits, dogs, baboons, cynomolgus monkeys, and humans. Thus in some preferred embodiments, the TLR9 agonist is an oligonucleotide comprising the sequence of SEQ ID NO:1.

Although the exemplary oligonucleotide TLR9 agonist, CpG 1018, is a CpG-ODN, the present disclosure is not restricted to fully DNA molecules. That is, in some embodiments, the TLR9 agonist is a DNA/RNA chimeric molecule in which the CpG(s) and the palindromic sequence are deoxyribonucleic acids and one or more nucleic acids outside of these regions are ribonucleic acids. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is circular or includes hairpin loop(s). The CpG oligonucleotide may be single stranded or double stranded.

In some embodiments, the CpG oligonucleotide may contain modifications. Modifications include but are not limited to, modifications of the 3′OH or 5′OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. Modified bases may be included in the palindromic sequence of the CpG oligonucleotide as long as the modified base(s) maintains the same specificity for its natural complement through Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the CpG oligonucleotide comprises a non-canonical base. In some embodiments, the CpG oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine. In some embodiments, the TLR9 agonist is an oligonucleotide comprising the sequence 5′-TCG₁AACG₁TTCG₁-3′ (SEQ ID NO:2), in which G₁ is 2′-deoxy-7-deazaguanosine. In some embodiments, the oligonucleotide comprises the sequence 5′-TCG₁AACG₁TTCG₁-X-G₁CTTG₁CAAG₁CT-5′, and in which G₁ is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).

The CpG oligonucleotide may contain a modification of the phosphate group. For example, in addition to phosphodiester linkages, phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester and phosphorodithioate and may be used in any combination. Other non-phosphate linkages may also be used. In some embodiments, the oligonucleotides comprise only phosphorothioate backbones. In some embodiments, the oligonucleotides comprise only phosphodiester backbones. In some embodiments, the oligonucleotide comprises a combination of phosphate linkages in the phosphate backbone such as a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides with phosphorothioate backbones can be more immunogenic than those with phosphodiester backbones and appear to be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of the present disclosure include at least one, two or three internucleotide phosphorothioate ester linkages. In some embodiments, when a plurality of CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, both stereoisomers of the phosphorothioate ester linkage are present in the plurality of CpG oligonucleotide molecules. In some embodiments, all of the internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages, or said another way, the CpG oligonucleotide has a phosphorothioate backbone.

A unit dose of the immunogenic composition, which is typically a 0.5 ml dose, may comprises from about 500 μg to about 5000 μg of the CpG oligonucleotide, preferably from about 750 μg to about 3000 μg of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises greater than about 500, 750, 1000, or 1250 μg of the CpG oligonucleotide, and less than about 3250, 3000, 2750, 2500, 2250, 2000, or 1750 μg of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 750, 1500, or 3000 μg of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 750 μg of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 1500 μg of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 3000 μg of the CpG oligonucleotide.

The CpG oligonucleotides described herein are in their pharmaceutically acceptable salt form unless otherwise indicated. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, zinc salts, salts with organic bases (for example, organic amines) such as N-Me-D-glucamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. In some embodiment, the CpG oligonucleotides are in the ammonium, sodium, lithium, or potassium salt form. In one preferred embodiment, the CpG oligonucleotides are in the sodium salt form.

B. SARS-CoV-2 Antigens

A SARS-CoV-2 antigen of the immunogenic compositions of the present disclosure comprises at least one SARS-CoV-2 protein or fragment thereof. In preferred embodiments, the SARS-CoV-2 antigen is recognized by SARS-CoV-2 reactive antibodies and/or T cells. In some embodiments, the SARS-CoV-2 antigen is an inactivated whole virus (COVID-19 virus). In other embodiments, the SARS-CoV-2 antigen is a subunit of the virus. In some embodiments, the SARS-CoV-2 antigen comprises a structural protein of SARS-CoV-2 or a fragment thereof. In some embodiments, the structural protein of SARS-CoV-2 comprises one or more of the group consisting of the spike (S) protein, the membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein. In some embodiments, the SARS-CoV-2 antigen comprises or further comprises a non-structural protein of SARS-CoV-2 or a fragment thereof. The nucleotide sequence of a representative SARS-CoV-2 isolate (Wuhan-Hu-1) is set forth as GenBank No. MN908947.3 (Wu et al., Nature, 579:265-269, 2020).

The amino acid sequence of a SARS-CoV-2 S protein is set forth as SEQ ID NO:4: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHV SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNENENGLTGTGVLTESNKKEL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLT PTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGI AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIG VTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNT FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVA KNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT. The signal peptide extends from residues 1-13, the extracellular region extends from residues 14-1213, the transmembrane domain extends from residues 1214-1236, and the cytoplasmic domain extends from residues 1237-1273.

In some preferred embodiments, the SARS-CoV-2 antigen comprises the receptor-binding domain (RBD) of the S protein, which is set forth as SEQ ID NO:5: NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ PYR. In some embodiments, the SARS-CoV-2 antigen comprises a variant of the RBD of the S protein having an amino acid sequence that it at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5. In some preferred embodiments, the SARS-CoV-2 antigen comprises the extracellular region of the S protein extending from residues 14-1213 of SEQ ID NO:4, or an amino acid sequence that it at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 14-1213 of SEQ ID NO:4. That is, in some embodiments, the SARS-CoV-2 antigen comprises a truncated, S protein devoid of signal peptide, transmembrane and cytoplasmic domains of a full length S protein. In some embodiments, the SARS-CoV-2 antigen is a recombinant protein, while in other embodiments, the SARS-CoV-2 antigen is purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.

A unit dose of the immunogenic composition, which is typically a 0.5 ml dose, may comprise from about 10 μg to about 100 μg of the SARS-CoV-2 antigen, preferably from about 25 μg to about 75 μg of the SARS-CoV-2 antigen, preferably from about 40 μg to about 60 μg of the SARS-CoV-2 antigen, or about 50 μg of the SARS-CoV-2 antigen. In some embodiments, the dose contains 5 μg of the SARS-CoV-2 antigen. In other embodiments, the dose contains 15 μg of the SARS-CoV-2 antigen. In further embodiments, the dose contains 25 μg of the SARS-CoV-2 antigen.

C. Additional Components

The immunogenic compositions of the present disclosure may comprise one or more additional components, such as one or more excipients, another adjuvant, and/or additional antigens.

1. Excipients

Pharmaceutically acceptable excipients of the present disclosure include for instance, solvents, bulking agents, buffering agents, tonicity adjusting agents, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments the immunogenic compositions may comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).

In some embodiments, the immunogenic compositions comprise an aqueous vehicle as a solvent. Suitable vehicles include for instance sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

The immunogenic compositions may comprise a buffering agent. Buffering agents control pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include for instance salts comprising acetate, citrate, phosphate or sulfate. Other suitable buffers include for instance amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7 or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of from about 6 to 9 in which the lower limit is less than the upper limit.

The immunogenic compositions may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include for instance dextrose, glycerol, sodium chloride, glycerin and mannitol.

The immunogenic compositions may comprise a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of degradation of the active agents during freeze or spray drying and/or during storage. Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

The immunogenic compositions may comprise a preservative. Suitable preservatives include for instance antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and is in a single use container, and thus does not necessitate inclusion of a preservative.

2. Additional Adjuvants

Adjuvants are known in the art and include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017). In some embodiments, the immunogenic compositions further comprises an aluminum salt adjuvant to which the SARS-CoV-2 antigen is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant consists of aluminum hydroxide. In some embodiments, a unit dose of the immunogenic composition, which is typically a 0.5 ml dose, comprises from about 0.25 to about 0.50 mg Al³⁺, or about 35 mg Al³⁺.

In other embodiments, the immunogenic composition further comprises an additional adjuvant. Other suitable adjuvants include, but are not limited to, squalene-in-water emulsion (e.g., MF59 or AS03), TLR3 agonists (e.g., poly-IC or poly-ICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such monophosphoryl lipid A (MPL), and/or a saponin such as Quil A or QS-21, as in AS01 or AS02), a TLR5 agonist (bacterial flagellin), and TLR7 and/or TLR8 agonists (imidazoquinoline derivatives such as imiquimod, and resiquimod)(Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, the additional adjuvant comprises MPL and alum (e.g., AS04). For veterinary use and for production of antibodies in non-human animals, mitogenic components of Freund's adjuvant (both complete and incomplete) can be used.

D. Kits

The present disclosure also provides kits comprising: i) an immunogenic composition comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide; and ii) a set of instructions for administration of the immunogenic composition to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof. Additionally, the present disclosure provides kits comprising: i) a first composition comprising a SARS-CoV-2 antigen; ii) a second composition comprising a TLR9 agonist, such as a CpG oligonucleotide; iii) instructions for mixing the first composition with the second composition to prepare an immunogenic composition; and optionally iv) a further set of instructions for administration of the immunogenic composition to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof. In some embodiments, the CpG oligonucleotide comprises the sequence 5′-AACGTTCG-3′. In some embodiments, the CpG oligonucleotide comprises the sequence 5′-AACGTTCGAG-3′ (SEQ ID NO:3). In some preferred embodiments, the CpG oligonucleotide comprises the sequence of 5′-TGACTGTGAA CGTTCGAGAT GA-3′ (SEQ ID NO:1).

The kits may comprise an immunogenic composition packaged appropriately. For example, if the immunogenic composition is a freeze-dried power, a vial with a resilient stopper is normally used so that the powder may be easily resuspended by injecting fluid (e.g., sterile water, saline, etc.) through the resilient stopper. In some embodiments, the kits comprise a device for administration (e.g., syringe and needle for intramuscular injection). The instructions relating to the use of the immunogenic composition generally include information as to dosage, schedule and route of administration for the intended methods of use.

II. Methods Of Use

The present disclosure relates to methods for stimulating an immune responses against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising: administering an immunogenic composition comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide, to a mammalian subject so as to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject. The immunogenic compositions of the present disclosure are intended for active immunization against COVID-19. In preferred embodiments, the immunogenic compositions are to be administered by intramuscular injection, optionally in a volume of about 0.5 mL (e.g., unit dose). In some embodiments, the intramuscular injection is into the deltoid muscle of the upper arm of a human subject in need thereof.

“Stimulating” an immune response, means increasing the immune response, which can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response includes but is not limited to one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating antibody production; stimulating interferon pathway-associated gene expression; stimulating chemoattractant-associated gene expression; and stimulating plasmacytoid dendritic cell (pDC) maturation. In some preferred embodiments, stimulating an immune response comprises increasing an antigen-specific antibody response in the subject.

ENUMERATED EMBODIMENTS

1. An immunogenic composition for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the SARS-CoV-2 antigen comprises a truncated, recombinant spike (S) protein devoid of signal peptide, transmembrane and cytoplasmic domains of a full length S protein, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject.

2. The composition of embodiment 1, wherein the oligonucleotide comprises the

sequence (SEQ ID NO: 3) 5′-AACGTTCGAG-3′

3. The composition of embodiment 1, wherein the oligonucleotide comprises the

sequence of (SEQ ID NO: 1) 5′-TGACTGTGAA CGTTCGAGAT GA-3′

4. The composition of any one of embodiments 1-3, wherein the oligonucleotide comprises a modified nucleoside, optionally wherein the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine.

5. The composition of embodiment 4, wherein the oligonucleotide comprises the sequence 5′-TCG₁AACG₁TTCG₁-3′ (SEQ ID NO:2) in which G₁ is 2′-deoxy-7-deazaguanosine, optionally wherein the oligonucleotide comprises the sequence 5′-TCG₁AACG₁TTCG₁-X-G₁CTTG₁CAAG₁CT-5′, in which G₁ is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).

6. The composition of any one of embodiments 1-5, wherein the oligonucleotide comprises at least one phosphorothioate linkage, or wherein all nucleotide linkages are phosphorothioate linkages.

7. The composition of any one of embodiments 1-6, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.

8. The composition of any one of embodiments 1-7, wherein a 0.5 ml dose of the immunogenic composition comprises from about 750 to about 3000 μg of the oligonucleotide, or wherein the immunogenic composition comprises about 750 μg, about 1500 μg, or about 3000 μg of the oligonucleotide.

9. The composition of any one of embodiments 1-8, wherein the SARS-CoV-2 antigen comprises a S protein ectodomain without a S1/S2 furin recognition site (e.g., RRAR), optionally wherein a 0.5 ml dose of the immunogenic composition comprises from about 5 to about 25 μg of the antigen, or wherein the immunogenic composition comprises about 5 μg, about 15 μg, or about 25 μg of the antigen.

10. The composition of embodiment 9, wherein the S protein ectodomain comprises the amino acid sequence of residues 14-1208 of SEQ ID NO:6 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% to residues 14-1208 SEQ ID NO:6.

11. The composition of embodiment 10, wherein the SARS-CoV-2 antigen is a fusion protein comprising a C-terminal trimerization domain.

12. The composition of embodiment 11, wherein the trimerization domain is a T4 fibritin trimerization domain, optionally comprising the amino acid sequence of SEQ ID NO:10 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:10.

13. The composition of any one of embodiments 1-12, wherein the SARS-CoV-2 antigen further comprises one or more of the SARS-CoV-2 membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein.

14. The composition of any one of embodiments 1-13, further comprising an aluminum salt adjuvant.

15. The composition of embodiment 14, wherein the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate 16. The composition of embodiment 14, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

17. The composition of any one of embodiments 14-16, wherein a 0.5 ml dose of the immunogenic composition comprises from about 0.25 to about 0.50 mg Al³±, or about 0.375 mg Al³±.

18. The composition of any one of embodiments 1-17, wherein the mammalian subject is a human subject.

19. A kit comprising:

-   -   i) the immunogenic composition of any one of embodiments 1-18,         and     -   ii) instructions for administration of the composition to         stimulate an immune response against the SARS-CoV-2 antigen in         the mammalian subject.

20. The kit of embodiment 19, further comprising iii) a syringe and needle for intramuscular injection of the immunogenic composition.

21. A method for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, comprising administering the immunogenic composition of any one of embodiments 1-18 to a mammalian subject so as to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.

22. The method of embodiment 21, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.

23. Use of the immunogenic composition of any one of embodiments 1-18 for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, the method comprising administering to the subject an effective amount of the immunogenic composition.

24. Use of the immunogenic composition of any one of embodiments 1-18 for protecting a mammalian subject from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the subject an effective amount of the immunogenic composition.

25. Use of the immunogenic composition of any one of embodiments 1-18 for preventing a mammalian subject from contracting COVID-19 disease, the method comprising administering to the subject an effective amount of the immunogenic composition.

26. The use of any one of embodiments 23-25, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.

EXAMPLES

Abbreviations: ACE2 (angiotensin-converting enzyme 2); alum (aluminum hydroxide); CHO cells (Chinese hamster ovary cells); CpG (unmethylated cytidine-phospho-guanosine); dpi (days post infection); ELISA (enzyme linked immunosorbent assay); IM (Intramuscular); mcg (micrograms); PFU (plaque-forming unit); RBD (receptor binding domain); RLU (relative luciferase units); SAE (serious adverse event); and SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2).

Example 1 Preparation of Recombinant SARS-CoV-2 Spike Protein Antigen and Immunogenic Compositions Thereof

A recombinant, stable prefusion SARS-CoV-2 spike protein (S-2P) was produced in Chinese hamster ovary (CHO) cells as described (see, Wrapp et al., Science, 367:1260-1263. 2020; Kuo et al., Scientific Reports, 10(1): 20085, 2020; and Lien et al., bioRxiv, 2021.01.07.425674. doi 10.1101/2021.01.07.425674). The S-2P protein was created by mutation of the S1/S2 furin-recognition site 682-RRAR-685 to GSAS and the 986-KV-987 site was mutated to PP. The amino acid sequence of the S-2P protein is set forth as SEQ ID NO:6:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLH STQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKS NIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHK NNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALH RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDIT PCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYS TGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGSASS VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTS VDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQ VKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGF IKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTI TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAI GKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI LSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTA PAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCD VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASV VNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ.

In order to produce the S-2P protein ectodomains, ExpiCHO-S cells (Thermo Fisher Scientific) were transfected using ExpiFectamine CHO transfection kit (Thermo Fisher Scientific) with a plasmid containing a mammalian-codon-optimized gene encoding the SARS-CoV-2 S-2P antigen fused in frame to a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an 8X His-tag and a Twin-Strep-tag (Wrapp et al., supra, 2020). The amino acid sequence of the T4 fibritin trimerization domain is set forth as SEQ ID NO:10: GYIPEAPRDGQAYVRKDGEWVLLSTF. The secreted S-2P protein was purified by affinity chromatography. Purification tags were subsequently removed by HRV3C protease digestion before the S-2P proteins were further purified. Under cryo-electron microscopy, the structure of the S-2P proteins was found to resemble typical SARS-CoV-2 spike trimers.

Immunogenic compositions comprising the S-2P protein were prepared by mixing the S-2P protein with either CpG 1018, aluminum hydroxide (alum), PBS, or CpG 1018 plus alum. The nucleic acid sequence of CpG 1018 (5′-TGACTGTGAA CGTTCGAGAT GA-3′) is set forth as SEQ ID NO:1. The immunogenic compositions were tested as vaccines in the preclinical and clinical studies described in Examples 2 and 3.

Example 2 Immunogenicity of CpG-Adjuvanted SARS-CoV-2 Subunit Vaccine in Mice and Hamsters

This example provides a description of preclinical studies to assess the immunogenicity of a SARS-CoV-2 subunit vaccine in mice and hamsters. The SARS-CoV-2 subunit vaccine is described in greater detail in Example 1.

A. Development of Adjuvanted Stable Prefusion SARS-CoV-2 Spike Protein Antigen

Materials and Methods

Pseudovirus production and titration. To produce SARS-CoV-2 pseudoviruses, a plasmid expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein was co-transfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica), using TranslT-LT1 transfection reagent (Minis Bio). Site-directed mutagenesis was used to generate the D614G variant by changing nucleotide at position 23403 (Wuhan-Hu-1 reference strain) from A to G. Mock pseudoviruses were produced by omitting the p2019-nCoV spike (WT). Seventy-two hours post-transfection, supernatants were collected, filtered, and frozen at −80° C. The transduction unit (TU) of SARS-CoV-2 pseudotyped lentivirus was estimated by using cell viability assay in response to the limited dilution of lentivirus. In brief, HEK-293 T cells stably expressing human ACE2 gene were plated on 96-well plate 1 day before lentivirus transduction. For the titering of pseudovirus, different amounts of pseudovirus were added into the culture medium containing polybrene. Spin infection was carried out at 1100×g in 96-well plate for 30 min at 37° C. After incubating cells at 37° C. for 16 hours, the culture media containing virus and polybrene were removed and replaced with fresh complete DMEM containing 2.5 μg/ml puromycin. After treating with puromycin for 48 h, the culture media were removed and cell viability was detected by using 10% AlarmaBlue reagents according to manufacturer's instruction. The survival rate of uninfected cells (without puromycin treatment) was set as 100%. The virus titer (transduction units) was determined by plotting the survival cells versus diluted viral dose.

Pseudovirus-based neutralization assay. HEK293-hAce2 cells (2×10⁴ cells/well) were seeded in 96-well white isoplates and incubated for overnight. Sera were heated at 56° C. for 30 min to inactivate complement and diluted in MEM supplemented with 2% FBS at an initial dilution factor of 20, and then twofold serial dilutions were carried out (for a total of 8 dilution steps to a final dilution of 1:5120). The diluted sera were mixed with an equal volume of pseudovirus (1000 TU) and incubated at 37° C. for 1 h before adding to the plates with cells. After the 1 h incubation, the culture medium was replaced with 50 μL of fresh medium. On the following day, the culture medium was replaced with 100 μL of fresh medium. Cells were lysed at 72 h post infections and relative luciferase units (RLU) were measured. The luciferase activity was detected by Tecan i-control (Infinite 500). The 50% and 90% inhibition dilution titers (ID₅₀ and ID₉₀) were calculated considering uninfected cells as 100% neutralization and cells transduced with only virus as 0% neutralization. Reciprocal ID₅₀ and ID₉₀ geometric mean titers (GMT) were both determined as ID₉₀ titers are useful when ID₅₀ titer levels are consistently saturating at the upper limit of detection.

Wild-type SARS-CoV-2 neutralization. The neutralization assay with SARS-CoV-2 virus was conducted as previously reported (Huang et al., J. Clin. Microbiol. 58(8): e01068-e1120, 2020). Vero E6 cells (2.5×10⁴ cells/well) were seeded in 96-well plates and incubated overnight. Sera were heated at 56° C. for 30 min to inactivate complement and diluted in serum-free MEM at an initial dilution factor of 20, and then further twofold serial dilutions were performed for a total of 11 dilution steps to a final dilution of 1:40,960. The diluted sera were mixed with an equal volume of SARS-CoV-2 virus at 100 TCID₅₀/50 μL (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession MT192759) and incubated at 37° C. for 2 h. The sera-virus mixture was then added to 96-well plate with Vero E6 cells and incubated in MEM with 2% FBS at 37° C. for 5 days. After incubation, cells were fixed by adding 4% formalin to each of the wells for 10 min and stained with 0.1% crystal violet for visualization. Results were calculated with the Reed-Muench method for log 50% end point for ID₅₀ and log 90% end point for ID % titers.

Immunization of mice. Female BALB/c and C57BL/6 mice were obtained from the National Laboratory Animal Center, Academia Sinica, Taiwan and BioLASCO Taiwan Co. Ltd. For antigen formulation, SARS-CoV-2 S-2P protein was mixed with either an equal volume of CpG 1018, aluminum hydroxide, PBS, or CpG 1018 plus aluminum hydroxide. Mice aged 6-9 weeks were immunized twice (50 μL intramuscularly in each of the left and right quadriceps femoris muscles per mouse) at 3 weeks apart as previously described (Pallesen et al., Proc. Natl. Acad. Sci. USA, 114(35): E7348-E7357, 2017). Total serum anti-S IgG and anti-RBD IgG titers were detected with direct ELISA using custom 96-well plates coated with S-2P antigen and an E. coli-expressed fragment of the S protein containing RBD region, respectively.

Cytokine assays. Two weeks after the second injection, mice were euthanized and splenocytes were isolated and stimulated with S-2P protein (2 μg/well) as previously described (Lu et al. Immunology, 130(2): 254-261, 2010). For detection of IFN-γ, IL-2, IL-4, and IL-5, the culture supernatant from the 96-well microplates was harvested to analyze the levels of cytokines by ELISA using Mouse IFN-γ Quantikine ELISA Kit, Mouse IL-2 Quantikine ELISA Kit, Mouse IL-4 Quantikine ELISA Kit, and Mouse IL-5 Quantikine ELISA Kit (R&D System). The OD450 values were read by Multiskan GO (Thermo Fisher Scientific).

Dose range finding study for single- and repeat-dose intramuscular injection (IM) in Sprague Dawley (SD) rats. Crl:CD Sprague Dawley (SD) rats were obtained from BioLASCO Taiwan Co. Ltd. Animal studies were conducted in the Testing Facility for Biological Safety, TFBS Bioscience Inc., Taiwan. SD rats aged 6-8 weeks were immunized with 5 μg, 25 μg or 50 μg of S-2P adjuvanted with either 1500 μg CpG 1018 alone or 750 μg CpG 1018 combined with 375 μg aluminum hydroxide. The test article or vehicle control was administered intramuscularly (0.25 mL/site, 2 sites of quadriceps femoris muscle) to each rat on Day 1 (for single-dose study) and Day 15 (for repeat-dose study). The observation period was 14 days (for single-dose study) and 28 days (for repeat-dose study). Parameters evaluated included clinical signs, local irritation examination, moribundity/mortality, body temperature, body weights, and food consumption during the in-life period. Blood samples were taken for hematology, including coagulation tests and serum chemistry. All animals were euthanized and necropsied for gross lesion examination, organ weights, and histopathology evaluation on injection sites and lungs.

Statistical analysis. For neutralization assays, geometric mean titers are represented by the heights of bars with 95% confidence intervals represented by the error bars. For cytokine and rat data, heights of bars or symbols represent means with SD represented by error bars. Dotted lines represent lower and upper limits of detection. Analysis package in Prism 6.01 (GraphPad) was used for statistical analysis. The data were compared at the same S-2P dose level with different adjuvant or at the same adjuvant system with varying antigen dose. Kruskal—Wallis with corrected Dunn's multiple comparisons test was used for non-parametric test between more than 2 experimental groups. Mann—Whitney U-test was used to compare two experimental groups. For correlation between antibody titers and neutralization titers, Spearman's rank correlation coefficient was used. *p<0.05, **p<0.01, ***p<0.001.

Results

Induction of potent neutralizing antibodies by CpG 1018 and aluminum hydroxide-adjuvanted S-2P. To facilitate establishment of stable clones for clinical studies and commercial production, the ExpiCHO system was used as the expression system of S-2P antigen. The S-2P proteins produced in CHO cells and their structure displayed typical spike trimers under cryo-EM, resembling that of 293-expressed SARS-CoV-2 S protein (Wrapp et al., Science, 367(6483): 1260-1263, 2020), suggesting that CHO cells are feasible in production of S-2P. Next, the potential of Th1-biasing CpG 1018 for clinical use was examined. Aluminum hydroxide (hereafter abbreviated as alum) was tested along with CpG 1018 since alum has been characterized to enhance the potency of CpG adjuvant when used in combination while also retaining the property of inducing Th1 responses (Thomas et al., Hum. Vaccin., 5(2): 79-84, 2009). The pseudovirus neutralization assay was performed with sera drawn either 3 weeks after the first injection or 2 weeks after the second injection. At 3 weeks after the first injection, neutralizing activities were already observed when mice were immunized with both 1 and 5 μg of S-2P with CpG 1018 and alum. At 2 weeks after the second injection, reciprocal inhibition dilution 50 (ID₅₀) GMT of 245, 3109, and 5120 were obtained with immunization of 1 μg S-2P adjuvanted with CpG 1018, alum, and with both CpG 1018 and alum, respectively (FIG. 1 ). Similar trends were observed at 5 μg of S-2P in both BALB/c and C57BL/6 mice.

Sera from these mice were then examined for the amount of anti-S IgG. CpG 1018 in combination with alum produced significantly higher titers of anti-S IgG compared to CpG 1018 alone (FIG. 2 ). To confirm the activities of the antibodies against the critical receptor-binding domain (RBD) of the S protein, immune sera were examined for anti-RBD IgG and the results were similar to that of the anti-S IgG with S-2P in combination with both CpG 1018 and alum induced the highest amount of IgG titer. There was a moderate correlation between anti-S IgG and anti-RBD IgG titers as shown by Spearman's rank correlation coefficient of 0.6486. The immune sera were further tested for their neutralization capabilities against wild-type SARS-CoV-2 in a neutralization assay. S-2P was able to inhibit SARS-CoV-2 at a concentration of 1 μg, although at lower potency than that of pseudovirus (FIG. 1 , FIG. 3 ). The reciprocal ID₅₀ GMT of 1 μg S-2P in the presence of CpG 1018, alum, and with both CpG 1018 and alum were approximately 60, 250, and 1500, respectively (FIG. 3 ). Pseudovirus carrying the current dominant D614G variant spike was also generated and neutralizing antibodies from mice immunized with S-2P with CpG 1018 and alum were effective against both pseudoviruses carrying the wild-type D614 and mutant D614G versions of spike proteins (FIG. 4 ). Neutralization titers of wild-type virus and pseudovirus and total anti-S IgG titers were all found to be highly correlated with Spearman's rank correlation coefficients greater than 0.8.

CpG 1018 induced Th1 immunity. To identify whether CpG 1018 could induce Th1 responses in the vaccine-adjuvant system, cytokines involved in Th1 and Th2 responses were measured in splenocytes from mice immunized with S-2P with alum, CpG 1018, or combination of the two. As expected, S-2P adjuvanted with alum induced limited amounts of IFN-γ and IL-2, the representative cytokines of Th1 response. In contrast, significant increases in IFN-γ and IL-2 were detected most strongly in high antigen dose plus CpG 1018 and alum. For Th2 response, while the levels of IL-4, IL-5 and IL-6 increased in the presence of alum and S-2P, addition of CpG 1018 to alum suppressed the levels of IL-5 and IL-6. IFN-γ/IL-4, IFN-γ/IL-5, and IFN-γ/IL-6 ratios are strongly indicative of a Th1-biased response and were increased by approximately 36-, 130-, and two-fold, respectively, in the presence of S-2P combined with CpG 1018 and alum (FIG. 5 ). These results suggested that the effect of CpG 1018 is dominant over alum in directing the cell-mediated response towards Th1 response, while retaining high antibody levels.

S-2P did not result in systemic adverse effects in rats. To elucidate the safety and potential toxicity of the vaccine candidate, 5 μg, 25 μg or 50 μg of S-2P adjuvanted with 1500 μg CpG 1018 or 750 μg CpG 1018 combined with 375 μg alum were administered to SD rats for single-dose and repeat-dose studies. No mortality, abnormality of clinical signs, differences in body weight changes, body temperature, nor food consumption were observed in either gender that could be attributed to S-2P (with or without adjuvant) with single dose administration. Increased body temperature at 4-h or 24-h after dosing was found in both genders of single-dose study and repeat-dose study; however, these temperature changes were moderate and were recovered after 48-h in both genders of all treated groups including controls (PBS). No gross lesions were observed in organs of most of the male and female rats with single-dose and two-dose administration, except for one male rat which was deemed to be non-vaccine-related. In conclusion, S-2P protein, with CpG 1018 or CpG 1018 with alum as adjuvants administrated intramuscularly once or twice to SD rats did not induce any systemic adverse effect.

SUMMARY

To develop a COVID-19 subunit vaccine, technology previously used for MERS-CoV was used to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. CpG 1018, a Th1-biasing oligonucleotide toll-like receptor 9 (TLR9) agonist was used as an adjuvant to enhance immunogenicity and mitigate potential vaccine-induced immunopathology. S-2P in combination with CpG 1018 and aluminum hydroxide (S-2P+CpG 1018+alum) was found to be a potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats receiving single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum.

B. Adjuvanted stable prefusion SARS-CoV-2 spike protein antigen provides protection from SARS-CoV-2 challenge

Materials and Methods

Pseudovirus-based neutralization assay and IgG ELISA. Lentivirus expressing the Wuhan-Hu-1 strain SARS-CoV-2 spike protein was constructed and the neutralization assay performed as previously described (Kuo et al., Scientific Reports, 10: 20085, 2020). Briefly, HEK293-hACE2 cells were seeded in 96-well white isoplates and incubated overnight. Sera from vaccinated and unvaccinated hamsters were heat-inactivated and diluted in MEM supplemented with 2% FBS at an initial dilution factor of 20, and then 2-fold serial dilutions were carried out for a total of 8 dilution steps to a final dilution of 1:5120. The diluted sera were mixed with an equal volume of pseudovirus (1,000 TU) and incubated at 37° C. for 1 hour before adding to the plates with cells. Cells were lysed at 72 hours post-infection and relative luciferase units (RLU) was measured. The 50% and 90% inhibition dilution titers (ID₅₀ and ID₉₀ were calculated referencing uninfected cells as 100% neutralization and cells transduced with only virus as 0% neutralization. Total serum anti-S IgG titers were detected with direct ELISA using custom 96-well plates coated with S-2P antigen.

Immunization and challenge of hamsters. Female golden Syrian hamsters aged 6-9 weeks old on study initiation were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The hamsters were randomized from different litters into four groups (n=10 for each group): hamsters were vaccinated intramuscularly with 2 injections of vehicle control (PBS), 1 or 5 μg of S-2P protein adjuvanted with 150 μg CpG 1018 and 75 μg aluminum hydroxide (alum), or adjuvant alone at 3 weeks apart. The hamsters were bled at 2 weeks after the second immunization via submandibular vein to confirm presence of neutralizing antibodies. Hamsters were challenged at 4 weeks after the second immunization with 1×10⁴ PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID Accession ID: EPI_ISL_411927) intranasally in a volume of 100 μL per hamster. The hamsters were divided into two cohorts to be euthanized on 3 and 6 days after challenge for necropsy and tissue sampling. Body weight and survival rate for each hamster were recorded daily after infection. On days 3 and 6 after challenge, hamsters were euthanized by carbon dioxide. The right lung was collected for viral load determination (RNA titer and TCID₅₀ assay). The left lung was fixed in 4% paraformaldehyde for histopathological examination.

Quantification of viral titer in lung tissue by cell culture infectious assay (TCID₅₀. The middle, inferior, and post-caval lung lobes of hamsters were homogenized in 600 μl of DMEM with 2% FBS and 1% penicillin/streptomycin using a homogenizer. Tissue homogenate was centrifuged at 15,000 rpm for 5 minutes and the supernatant was collected for live virus titration. Briefly, 10-fold serial dilutions of each sample were added onto Vero E6 cell monolayer in quadruplicate and incubated for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. The plates were washed with tap water and scored for infection. The fifty-percent tissue culture infectious dose (TCID₅₀)/mL was calculated by the Reed and Muench method (Reed and Muench, American Journal of Epidemiology, 27(3): 493-497, 1938).

Real-time RT-PCR for SARS-CoV-2 RNA quantification. To measure the RNA levels of SARS-CoV-2, specific primers targeting 26,141 to 26,253 region of the envelope (E) gene of SARS-CoV-2 genome were used in a TaqMan real-time RT-PCR method (Corman et al., Eurosurveillance. 25(3): 2000045, 2020). Forward primer E-Sarbeco-F1 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (SEQ ID NO:7) and the reverse primer E-Sarbeco-R2 5′-ATATTGCAGCAGTACGCACACA-3′ (SEQ ID NO:8), in addition to the probe E-Sarbeco-P1 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3′ (SEQ ID NO:9) were used. A total of 30 μL RNA solution was collected from each lung sample using RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. Five μL of RNA sample was added into a total 25 μL mixture of the Superscript III one-step RT-PCR system with Platinum Taq Polymerase (Thermo Fisher Scientific, USA). The final reaction mix contained 400 nM forward and reverse primers, 200 nM probe, 1.6 mM of deoxy-ribonucleoside triphosphate (dNTP), 4 mM magnesium sulfate, 50 nM ROX reference dye, and 1 μL of enzyme mixture. Cycling conditions were performed using a one-step PCR protocol: 55° C. for 10 mM for first-strand cDNA synthesis, followed by 3 min at 94° C. and 45 amplification cycles at 94° C. for 15 sec and 58° C. for 30 sec. Data was collected and calculated by Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). A synthetic 113-bp oligonucleotide fragment was used as a qPCR standard to estimate copy numbers of the viral genome. The oligonucleotides were synthesized by Genomics BioSci and Tech Co. Ltd. (Taipei, Taiwan).

Results

Hamsters as SARS-CoV-2 virus challenge model. To develop a SARS-CoV-2 virus challenge model in hamsters for the S2-P vaccine, an initial study was conducted to determine the optimal dose of virus for the challenge experiments. Unvaccinated hamsters were inoculated with 10³, 10⁴, or 10⁵ PFU of SARS-CoV-2 and euthanized on Day 3 or 6 after infection for tissue sampling. Following infection of 10³ to 10⁵ PFU of SARS-CoV-2, the hamsters exhibited dose-dependent weight loss. Hamsters infected with 10³ PFU gained weight while 10⁴ and 10⁵ PFU-infected hamsters experienced progressively severe weight loss at 6 days post-infection (dpi). However, there were no significant differences between levels of viral genome RNA and viral titer measured in 10³ to 10⁵ PFU of SARS-CoV-2-infected hamsters at 3 and 6 dpi All dosages of virus resulted in elevated lung pathology, even at 10³ PFU where the animals did not experience weight loss. There was also no virus inoculation dose-dependent effect on lung pathology scores and lung viral load. Therefore 10⁴ PFU of virus was used for virus challenge studies as it provides an adequate balance between clinical signs and virus titer for inoculation.

Administration of S-2P adjuvanted with CpG 1018 and aluminum hydroxide to hamsters induced high levels of neutralizing antibodies. Hamsters were divided into four groups receiving two immunizations at 21 days apart of either vehicle control (PBS only), adjuvant alone, low dose (LD) or high dose (HD) of S-2P+CpG 1018+alum. No differences in body weight changes were observed after vaccination among the four groups. Fourteen days after the second immunization, high level of neutralizing antibody titers were found in both LD and HD groups with ninety-percent inhibition dilution (ID₉₀) geometric mean titer (GMT) of 2,226 and 1,783, respectively (FIG. 6A). Anti-S IgG antibody levels were high enough that several individual samples reached the upper threshold of detection, with GMTs of LD and HD groups of U.S. Pat. Nos. 1,492,959 and 1,198,315, respectively (FIG. 6B). In general, even at a low dose, S-2P+CpG 1018+alum induced potent levels of immunogenicity in hamsters.

Adjuvanted S-2P protected hamsters from clinical signs and viral load after SARS-CoV-2 challenge. Four weeks after the second immunization, hamsters were challenged with 10⁴ PFU of SARS-CoV-2 virus and body weights were tracked up to 3 or 6 days post infection (dpi). Groups of animals were sacrificed on 3 or 6 dpi for viral load and histopathology analyses. LD and HD vaccinated groups did not show weight loss up to 3 or 6 days after virus challenge and instead gained 5 and 3.8 g of mean weight at 6 dpi, respectively. The protective effect was most significant at 6 dpi in both vaccinated groups, while vehicle control and adjuvant only groups experience significant weight loss. Lung viral load measured by viral RNA and TCID₅₀ assays showed that both viral RNA and viral titer decreased significantly at 3 dpi in vaccinated hamsters and dropped to below the lower limit of detection at 6 dpi (FIGS. 7A-7B). Note that viral load, especially viral titer measured by TCID₅₀ dropped noticeably at 6 dpi in control and adjuvant only groups due to hamsters' natural immune response (FIGS. 7A-7B). Lung sections were analyzed and pathology scoring was tabulated (FIG. 8 ). There were no differences at 3 dpi between control and experimental groups; however, at 6 dpi, the vehicle control and adjuvant only groups had significantly increased lung pathology including extensive immune cell infiltration and diffuse alveolar damage, compared to the HD antigen/adjuvant immunized groups (FIG. 8 ). These results showed that S-2P+CpG 1018+alum induced a robust immune response that was able to suppress viral load in lungs and prevent weight loss and lung pathology in infected hamsters.

SUMMARY

An adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, S-2P+CpG 1018+alum was applied to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of low dose or high dose S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to below the limit of detection in contrast to unvaccinated animals. Vaccination with either dose of adjuvanted S-2P produced comparable immunogenicity and protection from infection.

The significance of this study lies not only in the demonstration of in vivo efficacy, but also in safety. The viral challenge study allowed for the assessment of risk of disease enhancement with the vaccines. The histopathology scores of the immunized groups have not differed from the non-challenged animals, indicative of a lack of vaccine-enhanced pathology. Following the consensus made by CEPI and Brighton Collaboration in March 2020 (Lambert et al., Vaccine 38(31): 4783-4791, 2020), the animal study was run in parallel while a Phase I study was cautiously proceeding with careful review of safety data. The S-2P+CpG 1018+alum vaccines used in this study were from the same batch as the ones used in the Phase I study described in Example 3 (see ClinicalTrials.gov Identifier: NCT04487210).

Example 3 Immunogenicity of CpG-Adjuvanted SARS-CoV-2 Subunit Vaccine in Humans

This example provides a description of a Phase 1 study to be conducted in healthy, human subjects to assess safety and immunogenicity of a SARS-CoV-2 vaccine (see, NCT04487210). The SARS-CoV-2 subunit vaccine, which is referred to herein as “S-2P+CpG 1018+alum” or “MVC-COV1901”, is described in greater detail in Example 1.

Vaccines. MVC-COV1901 is formulated in with three different dosages of SARS-CoV-2 Spike (S) protein with CpG 1018 and aluminum hydroxide as adjuvants. Each vaccine contains 750 mcg CpG 1018 and 375 mcg aluminum and either 5 mcg, 15 mcg or 25 mcg of the S-2P protein antigen.

Objectives. The primary objective of this study is to evaluate the safety of MVC-COV1901 in three different strengths (5, 15, and 25 mcg S-protein with 750 mcg CpG 1018 and 375 mcg aluminum in the form of aluminum hydroxide as adjuvant) from Day 1 to 28 days after second vaccination. Secondary objectives include (1) evaluating the immunogenicity in terms of neutralizing antibody titers and binding antibody titers 14 days, 28 days after each vaccination, and 180 days after second vaccination; (2) evaluating the immunogenicity in terms of cellular immune responses 28 days after second vaccination and 180 days after second vaccination; and (3) evaluating the safety of MVC-COV1901 within the whole study period.

Study Design. This study is a phase I prospective, open-labeled, single-center study to evaluate the safety and immunogenicity of the SARS-CoV-2 vaccine MVC-COV1901. This study is a dose escalation study with three separate arms for subjects at the age of ≥20 and <50 years. The vaccination schedule consists of two doses of MVC-COV1901 for each study subject, administered by intramuscular (IM) injection 0.5 mL in the deltoid region of non-dominant arm preferably 28 days apart, on Day 1 and Day 29. In Phase 1a, 4 sentinel subjects are recruited to receive 5 mcg S-protein with adjuvant MVC-COV1901 to evaluate preliminary safety data of the vaccine. If no vaccine-related SAE occurs within 7 days after the first vaccination in the 4 sentinel subjects in Phase 1a, dosing of the remaining subjects in Phase 1a and Phase 1b proceeds. In Phase 1b, another 4 sentinel subjects are enrolled to receive 15 mcg S-protein with adjuvant MVC-COV1901 in Phase 1b. If no vaccine-related SAE occurs within 7 days after the first vaccination in the 4 sentinel subjects in Phase 1b, dosing of the remaining subjects in Phase 1b and Phase 1c proceeds. In Phase 1c, another 4 sentinel subjects are enrolled to receive 25 mcg S-protein with adjuvant MVC-COV1901. If no vaccine-related SAE occurs within 7 days after the first vaccination in the 4 sentinel subjects in Phase 1c, dosing of the remaining subjects in Phase 1c proceeds.

Study Population. Inclusion and exclusion criteria for study subjects include but are not limited to the listing provided below. Inclusion criteria include all of: male or female healthy volunteer ≥20 and <50 years of age; free of ongoing acute diseases or serious medical conditions (e.g. concomitant illness) such as cardiovascular, hepatic, psychiatric condition, medical history, physical findings, or laboratory abnormality that could interfere with the results of the trial or adversely affect the safety of the subject; female subject must be either of non-childbearing potential, or, if of childbearing potential, must be abstinent or agree to use medically effective contraception from 14 days before screening to 30 days following last injection of study vaccines; is willing and able to comply with all required study visits and follow-up required by this study; has no overseas travel within 14 days of screening and will not have any throughout the study period; and must provide written informed consent or the subject's legal representative must understand and consent to the procedure. Exclusion criteria include any one of the following: receiving any investigational intervention either currently or within 30 days of first dose; subject with previous known or potential exposure to SARS-CoV-1 or 2 viruses (except for those who have been tested negative and the 14-days self-managements/home quarantines/home isolations are completed), or received any other COVID-19 vaccine; administration of any vaccine within 4 weeks of first dose; a BMI greater than or equal to 30 kg/m²; has a history of hypersensitivity to any vaccine or a history of allergic disease or reactions likely to be exacerbated by any component of the MVC-COV1901; administration of any blood product or intravenous immunoglobulin administration within 12 weeks of first dose; pregnancy or breast feeding or have plans to become pregnant in 30 days after last injection of study vaccines; history of positive serologic test for HIV, hepatitis B surface antigen (HBsAg) or any potentially communicable infectious disease as determined by the investigator or Medical Monitor; positive serologic test for hepatitis C (exception: successful treatment with confirmation of sustained virologic response); baseline evidence of kidney disease as measured by creatinine greater than 1.5 mg/dL; screening laboratory tests with Grade 2 or higher abnormality (Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials, September 2007); immunosuppressive illness including hematologic malignancy, history of solid organ or bone marrow transplantation; history of autoimmune disease (systemic lupus, rheumatoid arthritis, scleroderma, polyarthritis, thyroiditis, etc.); current or anticipated concomitant immunosuppressive therapy (excluding inhaled, topical skin and/or eye drop-containing corticosteroids, low-dose methotrexate, or less than prednisone 20 mg/day or equivalent) within 12 weeks of first dose; current or anticipated treatment with TNF-α inhibitors, e.g. infliximab, adalimumab, etanercept within 12 weeks of first dose; prior major surgery or any radiation therapy within 12 weeks of first dose; alcohol or drug abuse or dependence, psychiatric, addictive, or any disorder that, in the opinion of the investigator, would interfere with adherence to study requirements or assessment of immunologic endpoints, or any illness or condition that in the opinion of the investigator may affect the safety of the participant or the evaluation of any study endpoint; presence of keloid scar formation or hypertrophic scar as a clinically significant medical condition, tattoos or wound covering the injection site area; body (oral, rectal or ear) temperature ≥38.0° C. or acute illness within 2 days of first dose, or acute respiratory illness within 14 days of first dose; screening laboratory test of antinuclear antibody (ANA), anti-dsDNA antibody, anti-neutrophil cytoplasmic antibodies (ANCA, including cytoplasmic ANCA, perinuclear ANCA) with the value higher than upper normal limit; or abnormal screening electrocardiography with clinically significant.

Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the examples should not be construed as limiting the scope of the disclosure, which is delineated by the appended claims. 

We claim:
 1. An immunogenic composition for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the SARS-CoV-2 antigen comprises a truncated, recombinant spike (S) protein devoid of signal peptide, transmembrane and cytoplasmic domains of a full length S protein, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject.
 2. The composition of claim 1, wherein the oligonucleotide comprises the sequence (SEQ ID NO: 3) 5′-AACGTTCGAG-3′.


3. The composition of claim 1, wherein the oligonucleotide comprises the sequence of 5′-TGACTGTGAA CGTTCGAGAT GA-3′(SEQ ID NO:1).
 4. The composition of claim 1, wherein the oligonucleotide comprises a modified nucleoside, optionally wherein the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine.
 5. The composition of claim 4, wherein the oligonucleotide comprises the sequence 5′-TCG₁AACG₁TTCG₁-3′ (SEQ ID NO:2) in which G₁ is 2′-deoxy-7-deazaguanosine, optionally wherein the oligonucleotide comprises the sequence 5′-TCG₁AACG₁TTCG₁-X-G₁CTTG₁CAAG₁CT-5′, in which G₁ is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).
 6. The composition of claim 3, wherein the oligonucleotide comprises at least one phosphorothioate linkage, or wherein all nucleotide linkages are phosphorothioate linkages.
 7. The composition of claim 6, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.
 8. The composition of claim 7, wherein a 0.5 ml dose of the immunogenic composition comprises from about 750 to about 3000 μg of the oligonucleotide, or wherein the immunogenic composition comprises about 750 μg, about 1500 μg, or about 3000 μg of the oligonucleotide.
 9. The composition of claim 8, wherein the SARS-CoV-2 antigen comprises a S protein ectodomain without a S1/S2 furin recognition site.
 10. The composition of claim 9, wherein the S protein ectodomain comprises the amino acid sequence of residues 14-1208 of SEQ ID NO:6 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% to residues 14-1208 of SEQ ID NO:6.
 11. The composition of claim 10, wherein the SARS-CoV-2 antigen is a fusion protein comprising a C-terminal trimerization domain.
 12. The composition of claim 11, wherein the trimerization domain is a T4 fibritin trimerization domain, optionally comprising the amino acid sequence of SEQ ID NO:10 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:10.
 13. The composition of claim 3, wherein the SARS-CoV-2 antigen further comprises one or more of the SARS-CoV-2 membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein.
 14. The composition of any one of claims 1-13, further comprising an aluminum salt adjuvant.
 15. The composition of claim 14, wherein the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.
 16. The composition of claim 14, wherein the aluminum salt adjuvant comprises aluminum hydroxide.
 17. The composition of claim 15, wherein a 0.5 ml dose of the immunogenic composition comprises from about 0.25 to about 0.50 mg Al³⁺, or about 0.375 mg Al³⁺.
 18. The composition of claim 17, wherein the mammalian subject is a human subject.
 19. A kit comprising: i) the immunogenic composition of claim 14, and ii) instructions for administration of the composition to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.
 20. The kit of claim 19, further comprising iii) a syringe and needle for intramuscular injection of the immunogenic composition.
 21. A method for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, comprising administering the immunogenic composition of claim 14 to a mammalian subject so as to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.
 22. The method of claim 21, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.
 23. Use of the immunogenic composition of claim 14 for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, the method comprising administering to the subject an effective amount of the immunogenic composition.
 24. Use of the immunogenic composition of claim 14 for protecting a mammalian subject from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the subject an effective amount of the immunogenic composition.
 25. Use of the immunogenic composition of claim 14 for preventing a mammalian subject from contracting COVID-19 disease, the method comprising administering to the subject an effective amount of the immunogenic composition.
 26. The use of any one of claims 23-25, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection. 